Vol. 56 No. 4/2009, 745–753
                                                                                                              on-line at: www.actabp.pl
                       Methods in Molecular Biology; vol. 498. High Throughput Protein 
                    Expression and Purification: Methods and Protocols, Sharon A. Doyle, 
                                ed. Humana Press 2009, pp 322, ISBN: 978-1-58829-879-9
                          The  end  of  the  twentieth  century  brought        a  short  summary  and  a  concise  theoretical  intro-
                  about  two  events  which  most  significantly  influ-        duction; it specifies all necessary materials and lists 
                  enced the further development of molecular biology.           numerous protocols which precisely explain how to 
                  The  first  was  the  sequencing  of  several  eukaryotic     perform each experiment. 
                  genomes, including the human one. The second was                     The  methodological  chapters  can  be  further 
                  the discovery of the RNAi phenomenon. The former              divided into two groups. The first one provides read-
                  had long been anticipated; the latter came as a sur-          ers with a wide spectrum of information on different 
                  prise.  Together, they drastically changed our think-         types of expression systems. Their key elements are 
                  ing  about  the  mechanisms  controlling  the  release        vectors into which one can insert any protein-coding 
                  and flow of genetic information from DNA through              sequence.  The  authors  have  decided  to  present  six 
                  RNA to proteins.  In  addition,  they  clearly  showed        frequently used systems which, as they believe, are 
                  that scientists have reached a point at which further         especially  well  designed  for  high  throughput  clon-
                  progress  in  our  understanding  of  complex  biologi-       ing,  clone  screening  and  gene  expression  in  E.  coli, 
                  cal systems requires that reductionist approaches be          yeast, insect and mammalian cells. By applying any 
                  replaced or complemented with more holistic ones.             of these systems one should be able rapidly to select 
                  Molecular biologists realized that the methods they           clones capable of producing a target protein in high 
                  were using to study individual genes, transcripts, or         amounts and in soluble form. Importantly, each of 
                  proteins are insufficient to investigate whole genom-         the presented systems offers a unique cloning strat-
                  es, transcriptomes, or proteomes.                             egy.  For  example, the Gateway system is based on 
                          One of  the  major  challenges  facing  modern        recombinational  cloning.  Flexi  vectors  require  liga-
                  molecular biology is to give a detailed description           tion-dependent cloning and in the case of In-Fusion 
                  of  cellular  proteomes.  Such  a  description  requires      and LIC vectors cloning is ligation-independent. 
                  not only that all proteins occurring in a given cell                 The  second  group  of  methodological  chap-
                  type  be  identified,  but  also  that  their  biochemical    ters presents various approaches used to improve 
                  properties and their structures be determined. The            the  efficacy  of  protein  production  in  prokaryotic, 
                  first stage on the long way to this goal is the elabo-        eukaryotic  or  cell-free  systems.  In  addition,  they 
                  ration of high throughput methods of protein pro-             show how applying specific protein tags, chroma-
                  duction  and  purification.  High  Throughput  Protein        tographic  techniques,  detergents,  or  other  chemi-
                  Expression and Purification: Methods and Protocols, ed-       cals  can  make  it  easier  and  faster  to  detect,  iso-
                  ited  by  Sharon A. Doyle, is an interesting attempt          late,  and  purify  target  proteins.  Special  attention 
                  showing what one currently has at one’s disposal              is  paid  to  so-called  “difficult  proteins”  which  re-
                  if  one wishes to make a first step toward this aim.          quire extraordinary techniques and methods to be 
                  The  book  is  divided  into  twenty  chapters  whose         expressed in sufficient amounts and proper form. 
                  authors  —  seventy-two  well-recognized  specialists         This  problem  concerns  primarily  highly  hydro-
                  — work at the best governmental laboratories and              phobic membrane proteins. 
                  private  companies,  all  of  them  global  leaders  in              Reading this review one can get the impres-
                  protein research.                                             sion  that  the  book  edited  by  Doyle  is  yet  another 
                          The  first  chapter  reviews  modern  strategies      handbook  of  protein  expression  and  purification, 
                  of  gene  cloning,  most effective  prokaryotic and eu-       similar to many others published in the past years. 
                  karyotic expression systems, and various methods of           One  crucial  factor,  however,  singles  it  out  from 
                  protein purification and analysis. The second chap-           among them. In the volume under review, the read-
                  ter shows how to design individual experiments and            ers  are  shown how to change the scale of their ex-
                  find an optimal method of protein production. The             periment from one clone or protein to hundreds of 
                  remaining  eighteen  (strictly  methodological)  chap-        them.  In  addition,  Doyle’s  book  helps  the  readers 
                  ters describe particular strategies or techniques that        learn how to apply the existing technologies, appa-
                  have been successfully applied for high throughput            ratus and robots to make automatic practically each 
                  protein  expression.  Each  of  these  chapters  contains     step of the protein production process: from cloning 
                                                                     Books reviews
               746                                                                                                                2009
               and clone screening, through protein expression and            also prove helpful to those who wish to optimize in-
               purification, to the analysis of the final products. Ac-       dividual protein expression, isolation or purification. 
               cordingly, this book should be recommended espe-                             Marek Figlerowicz
               cially  to  researchers  who  would  like  to  organize  a                   Institute of Bioorganic Chemistry 
               new laboratory focused on high throughput protein                            Polish Academy of Sciences 
               production,  or  to  improve  an  existing  one.  It  can                    Noskowskiego 12/14,  
                                                                                            61-704 Poznań, Poland 
                 Methods in Molecular Biology vol. 517, Toll-Like Receptors, Methods 
                  and Protocols, Claire E. McCoy and Luke A.J. O’Neill, eds. Series of 
                     Springer Protocols. Humana Press, 2009, ISBN: 978-1-934115-72-5
                       At  present  immunology belongs to the most            the  transcriptional  level,  expression  of  human  TIR 
               dynamically  developing  biomedical  sciences.  Re-            domains in bacteria, purification and crystallization, 
               cent  achievements  in  the  field  of  innate  immunity       tools  for  in  vitro  studies  of  ligands  and  host  cells 
               dramatically  have  changed  our  knowledge  of  the           with  the  use  of  commercially  available  ligands,  in-
               mechanisms involved in immune response. Discov-                vestigations of protein-protein interactions, subcellu-
               ery of Toll receptors in mid eighties ( awarded No-            lar localization and conformational changes in living 
               bel Prize in 1995) and further investigations resulted         cells  by  confocal  laser  scanning  microscopy  (LSM) 
               with the recognition of Toll-like receptors (TLR), al-         with GFP technology, fluorescence resonance energy 
               lowed  to  understand  how  immune  cells  recognize           transfer  (FRET),  bimolecular  fluorescence  comple-
               various  pathogen-associated  molecules  inducing  of          mentation (BiFC) and combination of confocal imag-
               both, innate and adaptive host defense response. To            ing with FRET. This part also includes data on bio-
               date ten human TLRs and thirteen mouse TLRs have               informatic  tools  for  analysis  of  TLR  sequences  and 
               been  identified.  TLRs  contain  leucine-reach  repeats       three-dimensional  structures,  as  well  as  for  predic-
               (LRR) in their extra cellular domain and an intrac-            tion and characterization of ligand binding. Experi-
               ellular region that share homology with interleukin-           mental procedures for measurements of TLR interac-
               1  receptor  called  TIR.  Upon  receptor  activation  the     tions with their ligands are also described.
               TIR domain recruits downstream adaptor signaling                      The second part consists chapters on methods 
               molecules i.e. protein kinases that activate transcrip-        of analysis of the TLR down-stream signal transduc-
               tion factors NFκB or IRFs and trigger specific genes           tion  pathways.  Key  intermediates  of  the  signaling 
               expression responsible for realization of the effector         pathways are identified after expression in bacteria, 
               function of the cell. The importance of TLRs in im-            by  liquid  chromatography  and  tandem  mass  spec-
               mune response and their implications in pathogen-              trometry (LC-SM/SM) or by differential in-gel elec-
               esis  of  various diseases are the subject of many in-         trophoresis  DIGE  (2D  electrophoresis  coupled  with 
               vestigations. Further understanding of the molecular           protein  labeling)  using  proteomic  strategies.  The 
               mechanisms  of  TLR  signal  transduction  pathways,           mammalian  protein-protein  interaction  trap  (MAP-
               involving  receptor-ligand  interactions  and  cascades        PIT)  technique  was  used  to  study  early  signaling 
               of  intracellular  signaling  including  posttranslational     steps from TLR4 to IRAK-1. The methodological ap-
               modifications, is crucial to reveal its biological role        proaches used to analyze the role of posttranslation-
               in  health  and  disease.  This  is  possible  only  by  ap-   al  modifications  (phosphorylation,  ubiquitination) 
               plication and development of many analytical tech-             on TLR signaling pathways include transient trans-
               niques and interdisciplinary studies combining new             fections,  site-directed  mutagenesis,  immunoprecipi-
               achievements in the fields of molecular biology, ge-           tation and immunobloting . Techniques for study of 
               netics, biochemistry and immunology.                           TLR stimulation on apoptosis and negative regula-
                       The book “Toll-Like Receptors: Methods and             tion  of  TLR  signaling,  including  influence  of  viral 
               Protocols”  with  65  contributors,  focuses  on  recent       proteins, are also described.
               developments  of  TLRs  investigations  performed                     The  third  part  describes  genetic  techniques 
               by  many  institutions  including  Universities,  Tech-        applied in TLR analysis. A practical guide is given 
               nical  Universities  and  Medical  Schools  in  Europe         for performing germline mutagenesis in mouse using 
               and USA. It comprise 25 chapters (447 pages) with              ENU (N-ethyl-N-nitrosurea) for identification of the 
               black-  and-  white  illustrations  and  color  plates,  di-   genes involved in TLR signaling. Receptor functions 
               vided into four parts covering the different aspects           are  investigated  by  microarray  techniques  using 
               of TLRs studies.                                               Two-Color  Microarray  assay.  Microarray  process-
                       The  first  part  is  focused  on  methods  of  de-    ing and data analysis are described, including list of 
               tection  and  analysis  of  TLRs  including  analytical        software and web sites. Two very important new ap-
               RT-PCR  approach  for  study  of  TLR  expression  at          proaches are described. One is application of siRNA 
                                                                         Books reviews
                   Vol. 56                                                                                                             747
                   technology, a superior method of post-transcription-          tive  gene  expression  measurement  assay  based  on 
                   al  gene silencing (PTGS) for uncovering novel gene           PCR technique with biomarkers of the TLR activity, 
                   functions in TLR signaling. Second is the discovery           and its applications in clinical trials, presented also 
                   by  genotypic  methods  of  TLR  polymorphism,  that          in  this  part  ,  will  allow  development  of  innovative 
                   affects  host  susceptibility  towards  infections,  RLFP,    therapies.
                   genotyping  via  the  Sequenom  and  Illumina  plat-                  All chapters provide short introduction, step-
                   forms and direct sequencing are p[resented.                   by-  step  laboratory  protocols  with  lists  of  materi-
                          The four part describes techniques applied to          als  and reagents and, what is very important, their 
                   study of  the  role  of  TLRs  in  various  diseases  with    sources. Experimental tips and troubleshooting pro-
                   use of experimental models of pathogen-host in vitro          cedures  are  also  included,  which  is  invaluable  for 
                   and  in  vivo  interactions.  It  involves  investigations    beginners in the field. 
                   of animal responsiveness to systemic challenge, iso-                  The covering of such wide range of problems 
                   lated  and  cultured  cell  activation  and  proliferation    concerning investigations of the role of TLRs in the 
                   assays, reporter gene activation assay and immuno-            innate and adaptive immune response makes the re-
                   histochemistry (IHC) with immunofluorescence (IF).            viewed book an excellent manual for all investiga-
                   The  presented  data  include  experimental  disease          tors  as  well  as  teachers  and  advanced  students  of 
                   models of acute infection, septic shock and athero-           biological and medical sciences interested in modern 
                   sclerosis,  and  in  vitro  investigations  of  cultured  fi- immunology. That book fills an informational gap in 
                   broblasts from rheumatoid arthritis patients and tis-         very vital field of knowledge.
                   sue  samples  of  patients  with  collateral  cancer.  The                   Elżbieta Wałajtys-Rode
                   involvement of TLRs in adaptive immune response                              Department of Biochemistry and Biotechnolo-
                   is documented by studies of the activation of mouse                          gy, Chemical Faculty,  
                   B cells and dendritic cells by immune complexes (IC)                         Rzeszow University of Technology 
                   characteristic of autoimmune diseases. The quantita-                         Al. Powstańców Warszawy 6, 
                                                                                                35-959 Rzeszów, Poland
                         Methods in Molecular Biology; vol. 541. Computational Systems 
                       Biology, Jason McDermott, Ram Samudrala, Roger E. Bumbgarner, 
                            Kristina Montgomery, Reneé Ireton, eds. Humana Press 2009.
                          The new volume of the Humana Press “Meth-              Then, every base pair in DNA is changed to another 
                   ods in Molecular Biology” series, entitled “Compu-            possible base pair. Molecular dynamics and free en-
                   tational  Systems  Biology,”  consists  of  25  chapters      ergy calculations are used to obtain the relative free 
                   authored by 57 specialists in the field. As is noted in       energies of binding to different sequences which can 
                   one of these chapters (Chapter 20), a one-gene-one-           then be combined to a single position weight matrix. 
                   study approach is unlikely to enable the identifica-          The next chapters are dedicated to the inference of 
                   tion  of  the  functions  of  all  genes/products  and  un-   protein-protein  interactions:  using  known  interac-
                   derstanding of  a given cell or organism as a whole.          tions  after  a  careful  choice  of  protein  domains  that 
                   High-throughput approaches bring this goal closer,            explain the interactions (Chapter 3) or correlation of 
                   and  the  huge  amounts  of  data  generated  require         evolutionary divergence (Chapter 4, with an impor-
                   the development of new computational methods to               tant  conclusion  that  the  co-evolution  model  poorly 
                   handle it and to use it to build useful models. This          predicts  protein-protein  interactions).  Chapter  5  is 
                   book contains a wide collection of methods that can           more general and reviews algorithms used to inte-
                   be  used  with  this  goal  in  mind  and  gives  a  broad    grate  genome  data  to  predict  protein-protein  inter-
                   review  of  this  fascinating  and  quickly  developing       actions using machine learning. It gives some com-
                   field.                                                        ments on the interaction data sets: for example, on 
                          Part  I  of  the  book  (“Network  Components”)        the  bias  towards  important  proteins  and  important 
                   starts with methods that are related to finding edg-          interactions in the positive interaction set and prob-
                   es  linking  nodes  in  regulatory  networks.  Chapter        lems with constructing a negative data set (a set of 
                   1  presents  a  quite  detailed  protocol  of  how  to  use   proteins  that  are  known  not  to  interact).  The  next 
                   a  specific  software  package  (A-GLAM)  to  identify        chapter (Chapter 6) is again a very detailed descrip-
                   cis-regulatory elements from the combination of co-           tion of a method to use the information on transcrip-
                   expression  data  and  functional  annotations.  Chap-        tional regulation in a model organism to infer regu-
                   ter  2  provides  a  different  perspective:  a  method  to   latory interactions in a related organism. The proce-
                   find  transcription  factor  binding  sites  based  on  the   dure starts from finding similar gene pairs using se-
                   3-dimensional  structures  of  transcription  factors         quence search. The predicted edges in the regulatory 
                   complexed with DNA (the starting point structures             network are then filtered by the search for binding 
                   can be determined experimentally or by modelling).            sites,  functional annotation, protein localisation, etc. 
                                                                        Books reviews
                748                                                                                                                   2009
                The last chapter in Part I (Chapter 7) stands out as            fluctuations in the concentration of biomolecules. It 
                the only one that discusses metabolic networks. Not             provides an extensive list of software tools for kinet-
                only does it provide an exceptionally accessible in-            ic simulations of systems. The last chapter of Part III 
                troduction to the analysis of general network prop-             (Chapter 15) would be of interest to molecular biolo-
                erties,  but  also  describes  a  very  interesting  way  to    gists that might wish to apply their skills to provid-
                reveal  the  hierarchy  in  biological  networks.  This  is     ing  the  computational  biologists  with  high  quality 
                also  one  of  two  chapters  in  the  book  accompanied        data. As an example, the authors describe their own 
                by colour plates.                                               study  of  promoter  regulation  in  phage  λ  in  which 
                       The  difference  of  focus  between  Part  I  and        computational  modelling  allowed  them  to  measure 
                Part II (“Network Inference”) of the book is hardly             the energetics of DNA looping. 
                noticeable.  Chapter  8  considers  again  the  problem                 Part IV (“Function and Evolutionary Systems 
                of  inferring  transcriptional  regulatory  networks  in        Biology”)  starts with a chapter presenting a model 
                non-model  organisms.  A  simple  approach  is  used:           of intron evolution (Chapter 16). The authors derive 
                if  a  transcription factor and its target gene have or-        very  interesting  conclusions  from  their  analysis  (p. 
                thologs in the non-model genome, an interaction is              360), for example, that the frequencies of intron gain 
                inferred. The authors consider significance of conser-          and loss are correlated (suggesting a common mech-
                vation  of  genes,  interactions,  network  motifs  (such       anism for both processes) and that the frequency of 
                as feed-forward motif etc.) and global network struc-           intron gain is correlated with gene expression level. 
                ture. It may be slightly disappointing that results are         Sadly, the results themselves are not presented; the 
                discussed only cursorily in Note 11 (p. 178). Chapter           bulk of the chapter is dedicated to a very detailed 
                9 provides a general introduction to the problem of             analysis of the algorithm. Chapter 17 is dedicated to 
                inference of transcriptional networks from data ob-             functional prediction (with an example of prediction 
                tained using microarrays, protein-DNA and protein-              of the EC number and Pfam classification) from fea-
                protein  interaction  assays,  and  functional  annota-         tures extracted from sequences (such as amino acid 
                tions.  It  then  proceeds  to  describe  methods,  giving      composition, average hydrophobicity, protein length 
                details on the authors’ own tool, Inferelator. The in-          etc.)  and  data  such  as  tissue  specificity,  subcellular 
                troduction to Chapter 10 starts with an observation             localisation,  cofactors  etc.  The  authors’  algorithm 
                that differential gene expression is sometimes linked           uses mixtures of stochastic decision trees with nov-
                to  variation  in  specific  genomic  locations;  methods       el  criterion  functions  to  evaluate  tree  performance. 
                to  find  regulatory  pathways  from  such  expression          The next chapter (Chapter 18) presents a method to 
                quantitative  trait  loci  are  first  outlined,  then  evalu-  identify  specificity-determining  residues  (in  brief, 
                ated using gene knockout data. The subject of reg-              conserved  in  orthologs  but  different  in  paralogs) 
                ulatory  network  inference  is  continued  in  the  last       and residues co-evolving in interacting proteins. An 
                chapter  of  Part  II  (Chapter  11),  with  emphasis  on       example  of  identifying  such  residues  in  bacterial 
                Bayesian networks as an approach to obtaining net-              two-component  (histidine  kinase/response  protein) 
                works  with  predictive  value  from  expression  data          systems is provided. Colour plates are used to illus-
                and other data types.                                           trate  the  results.  Chapter  19  describes  how  to  take 
                       In  Part  III  (“Network  Dynamics”),  the  focus        advantage of the information about protein-protein 
                slowly shifts. Chapter 12 describes in intricate detail         interactions to understand clinical phenotypes: genes 
                two algorithms: CoExMiner, capable of handling not              coding  for  proteins  interacting  with  known  dis-
                only linear, but also nonlinear relationships of gene           ease-associated proteins are better candidates when 
                co-expression  and  of  suggesting  directionality  of          searching  for  disease-causing  mutations  among 
                regulatory relationships, and PathwayPro, a tool to             polymorphisms. A method to discriminate between 
                simulate the effects of perturbations to the network            damaging and neutral mutations using support vec-
                (such  as  a  change  of  one  or  several  gene  expres-       tor machines is presented. Chapter 20 discusses the 
                sions) on its complex behaviour, as a way to identi-            problem of functional bias in gene annotations. Such 
                fy targets for therapeutic intervention. Chapter 13 is          bias  can  affect  further  discovery  of  gene  functions 
                even  more  mathematically  advanced  and  describes            from expression data. For example, out of more than 
                the  theory  to  analyse  the  dynamics  of  biochemi-          1000 functional annotation terms, one (‘protein bio-
                cal  networks  (biochemical  control  theory)  with  ap-        synthesis’ ) covers more than 4% of annotations. The 
                plications to some basic motifs in cellular networks            authors observed that removing proteins pairs shar-
                (e.g.  branches  or  feedback  loops),  with  important         ing this term resulted in lower correlation between 
                conclusions  for  those  wishing  to  target  pathways.         the likelihood of functional coupling and co-expres-
                The  chapter  ends  with  a  comprehensive  reading             sion, while the correlation between the likelihood of 
                list.  While Chapter 13 concentrates on deterministic           functional coupling and the measure of protein-pro-
                models, Chapter 14 is somewhat complementary, as                tein  interaction  remained  unaffected.  They  propose 
                it  introduces  the  approaches  to  address  stochastic        that  masking  dominant  terms  can  help  when  ma-